Conceptually, pseudo-complementary DNA (pcDNA) is a "structure-free" polymer composed of base analogs that don't interact with each other but are able to Watson-Crick pair to regular bases in a complementary probe. Base pairs in the resulting hybrids are composed of unique combinations of modified and natural nucleotides. The ability to convert native DNA or RNA into structure-free pcDNA would significantly improve the reliability and efficiency of short probes by eliminating interference from secondary structure in the target. The exquisite ability of such probes to discriminate against mismatches favors their use in the direct detection of single nucleotide polymorphisms. pcDNA targets would also facilitate the practical use of universal oligonucleotide microarrays and the development of extremely dense SNP microarrays. High throughput sequencing technologies based on oligonucleotide ligation or passage through a nanopore would also benefit. Recently, we developed a set of dNTP analogs that support enzymatic synthesis of DNA with reduced secondary structure and increased accessibility to short unmodified probes. In this grant application we propose to optimize methods for the preparation of pcDNA and to rigorously characterize the hybridization properties of this DNA with respect to accessibility, stability and specificity. Significant effort will be expended in developing a new, rapid and cost-effective approach to determining nearest neighbor free energies for both pairing and mis-pairing of novel bases such as the ones used to prepare pcDNA. This approach will use a microarray platform to simultaneously determine the melting profiles of hundreds of short duplexes. Successful completion of this research program will provide a comprehensive analysis of the practicality and benefits of pseudo-complemementary nucleic acids and establish whether microarrays can be used to simplify and accelerate the acquisition of free energy parameters for hybridization. PUBLIC HEALTH RELEVANCE: Short pieces of nucleic acid known as oligonucleotides have applications in human diagnostics, pharmacogenetics and high throughput sequencing due to their ability to interact with DNA and RNA. Unfortunately, the performance of oligonucleotides is impaired by the presence of higher order structure in naturally occurring DNA and RNA. In this grant we will evaluate a novel strategy for eliminating higher order structure in DNA thereby improving the utility of oligonucleotides for both new and existing applications. In reducing the technology to practice, we will test a new microarray based strategy for acquiring the thermodynamic parameters that describe the interaction of oligonucleotides with "structure-free" DNA. This thermodynamic library will facilitate use of the new technology.